There is an urgent need to develop safer smallpox vaccines. One approach is the use of modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia virus that does not replicate in human cells. In comparison to vaccinia virus, however, MVA is less immunogenic and may require higher doses and more frequent boosts. There also remain safety concerns about the use of a live attenuated virus and the potential presence of adventitious pathogens. Alternative approaches include the development of inactivated virus or recombinant protein vaccines. However, vaccine development is hampered because little is known about what proteins play an important role in the generation of protective immune responses to smallpox. Recently, 4 envelope proteins that induce neutralizing antibodies were identified, and DNA vaccination with a combination of all 4 genes protected mice from a lethal vaccinia infection. Although a vaccine will need to induce strong T cell responses, in addition to neutralizing antibodies, no information is available about the vaccinia protein targets recognized by T cells. In particular, CD4 T cells are required to enhance B cell, T cell, and innate immune responses to lytic viruses. As a first step, peripheral blood mononuclear cells from healthy adults previously immunized with vaccinia virus will be tested for virus-specific T cell responses. The goals of this study are: 1) to measure the frequencies of human T cell responses to vaccinia structural proteins; 2) to identify major CD4 and CD8 T cell epitopes within the above 4 envelope proteins; and 3) to analyze the frequencies of the T cell responses to these epitopes post-vaccination. It is anticipated that this work will provide new tools for the detection and monitoring of vaccinia-specific T cell responses from most individuals and will help develop new strategies for smallpox vaccine development.